Advances in umbilical cord blood cell therapy: the present and the future.

Umbilical cord blood (UCB), previously seen as medical waste, is increasingly recognized as a valuable source of cells for therapeutic use. The best-known application is in hematopoietic stem cell transplantation (HSCT), where UCB has become an increasingly important graft source in the 28 years since the first umbilical cord blood transplantation (UCBT) was performed. Recently, UCB has been increasingly investigated as a putative source for adoptive cell therapy.

Microwell-Based Live Cell Imaging of NK Cell Dynamics to Assess Heterogeneity in Motility and Cytotoxic Response.

NK cell heterogeneity has primarily been studied either on the population level, measuring average responses, or on the single cell level by flow cytometry, providing static snapshots. These approaches have certain drawbacks, not enabling dynamic observations of single cells over extended periods of time. One of the primary limitations of single cell imaging has been throughput; it has been challenging to collect data for many cells due to their dynamic nature and migrating out of the field of view.

Microchip Screening Platform for Single Cell Assessment of NK Cell Cytotoxicity.

Here, we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK) cells within larger populations. Human primary NK cells were distributed across a silicon-glass microchip containing 32,400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis, the numbers of NK and live or dead target cells in each well could be assessed at different time points after initial mixing.

Distinct Migration and Contact Dynamics of Resting and IL-2-Activated Human Natural Killer Cells.

Natural killer (NK) cells serve as one of the first lines of defense against viral infections and transformed cells. NK cell cytotoxicity is not dependent on antigen presentation by target cells, but is dependent on integration of activating and inhibitory signals triggered by receptor-ligand interactions formed at a tight intercellular contact between the NK and target cell, i.e.

Live cell imaging in a micro-array of acoustic traps facilitates quantification of natural killer cell heterogeneity.

Natural killer (NK) cells kill virus-infected or cancer cells through the release of cytotoxic granules into a tight intercellular contact. NK cell populations comprise individual cells with varying sensitivity to distinct input signals, leading to disparate responses. To resolve this NK cell heterogeneity, we have designed a novel assay based on ultrasound-assisted cell-cell aggregation in a multiwell chip allowing high-resolution time-lapse imaging of one hundred NK-target cell interactions in parallel.

Classification of human natural killer cells based on migration behavior and cytotoxic response.

Despite intense scrutiny of the molecular interactions between natural killer (NK) and target cells, few studies have been devoted to dissection of the basic functional heterogeneity in individual NK cell behavior. Using a microchip-based, time-lapse imaging approach allowing the entire contact history of each NK cell to be recorded, in the present study, we were able to quantify how the cytotoxic response varied between individual NK cells. Strikingly, approximately half of the NK cells did not kill any target cells at all, whereas a minority of NK cells was responsible for a majority of the target cell deaths.

Analysis of transient migration behavior of natural killer cells imaged in situ and in vitro.

We present a simple method for rapid and automatic characterization of lymphocyte migration from time-lapse fluorescence microscopy data. Time-lapse imaging of natural killer (NK) cells in vitro and in situ, both showed that individual cells transiently alter their migration behavior. Typically, NK cells showed periods of high motility, interrupted by transient periods of slow migration or almost complete arrests.

A silicon-glass microwell platform for high-resolution imaging and high-content screening with single cell resolution.

We present a novel microwell array platform suited for various cell-imaging assays where single cell resolution is important. The platform consists of an exchangeable silicon-glass microchip for cell biological applications and a custom made holder that fits in conventional microscopes. The microchips presented here contain arrays of miniature wells, where the well sizes and layout have been designed for different applications, including single cell imaging, studies of cell-cell interactions or ultrasonic manipulation of cells.

Imaging immune surveillance of individual natural killer cells confined in microwell arrays.

New markers are constantly emerging that identify smaller and smaller subpopulations of immune cells. However, there is a growing awareness that even within very small populations, there is a marked functional heterogeneity and that measurements at the population level only gives an average estimate of the behaviour of that pool of cells. New techniques to analyze single immune cells over time are needed to overcome this limitation.

Ultrasound-controlled cell aggregation in a multi-well chip.

We demonstrate a microplate platform for parallelized manipulation of particles or cells by frequency-modulated ultrasound. The device, consisting of a silicon-glass microchip and a single ultrasonic transducer, enables aggregation, positioning and high-resolution microscopy of cells distributed in an array of 100 microwells centered on the microchip. We characterize the system in terms of temperature control, aggregation and positioning efficiency, and cell viability.

Live cell linear dichroism imaging reveals extensive membrane ruffling within the docking structure of natural killer cell immune synapses.

We have applied fluorescence imaging of two-photon linear dichroism to measure the subresolution organization of the cell membrane during formation of the activating (cytolytic) natural killer (NK) cell immune synapse (IS). This approach revealed that the NK cell plasma membrane is convoluted into ruffles at the periphery, but not in the center of a mature cytolytic NK cell IS. Time-lapse imaging showed that the membrane ruffles formed at the initial point of contact between NK cells and target cells and then spread radialy across the intercellular contact as the size of the IS increased, becoming absent from the center of the mature synapse.

NK cell-mediated lysis is essential to kill Epstein-Barr virus transformed lymphoblastoid B cells when using rituximab.

Rituximab is a humanized chimeric monoclonal antibody, targeted against the pan B cell marker CD20. It is frequently used to treat a variety of B cell lymphomas and immunosuppression associated lymphoproliferations such as posttransplant lymphoproliferative disorder (PTLD). The response rate of rituximab treatment is 65%, but the exact in vivo mechanism of action is not yet fully understood, although antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and direct induction of apoptosis have been suggested as effector mechanism.

Effect of frequently used chemotherapeutic drugs on the cytotoxic activity of human natural killer cells.

Tumors are considered to be possible targets of immunotherapy using stimulated and expanded autologous or allogeneic natural killer (NK) cells mismatched for MHC class I molecules and inhibitory NK receptors. NK cell-based immunoadjuvant therapies are carried out in combination with standard chemotherapeutic protocols. In the presented study, we characterized the effect of 28 frequently used chemotherapeutic agents on the capacity of NK cells to kill target cells.

Human and murine inhibitory natural killer cell receptors transfer from natural killer cells to target cells.

Intercellular transfer of proteins across the immunological synapse is emerging as a common outcome of immune surveillance. We previously reported that target-cell MHC class I protein transfers onto natural killer (NK) cells expressing cognate killer Ig-like receptors (KIRs). We now show that, for both murine and human cells, target cells expressing inhibitory MHC class I ligands acquire cognate inhibitory NK receptors.

The protean immune cell synapse: a supramolecular structure with many functions.

Heterogeneity in the supramolecular organization of immunological synapses arises from the involvement of different cells, distinct environmental stimuli, and varying levels of protein expression. There may also be heterogeneity in the types and amounts of cell surface proteins and lipids that transfer between lymphocytes during immune surveillance. In addition, immune cells can be involved in the assembly of a 'viral synapse', such that micrometer-scale organization of proteins at intercellular contacts occurs during transmission of a virus between T cells.

The size of the synaptic cleft and distinct distributions of filamentous actin, ezrin, CD43, and CD45 at activating and inhibitory human NK cell immune synapses.

In this study, we report the organization of cytoskeletal and large transmembrane proteins at the inhibitory and activating NK cell immunological or immune synapse (IS). Filamentous actin accumulates at the activating, but not the inhibitory, NK cell IS. However, surprisingly, ezrin and the associated protein CD43 are excluded from the inhibitory, but not the activating, NK cell IS.

Imaging immune surveillance by T cells and NK cells.

As T cells and natural killer (NK) cells survey the surface of other cells, cognate receptors and ligands are commonly organized into distinct micrometer-scale domains at the intercellular contact, creating an immune or immunological synapse (IS). We aim to address the still unanswered questions of how this organization of proteins aids immune surveillance and how these domains are biophysically constructed. Molecular mechanisms for the formation of the IS include a role for the cytoskeleton, segregation of proteins according to the size of their extracellular domains, and association of proteins with lipid rafts.